cellular/molecular the p75 neurotrophin receptor …cellular/molecular the p75 neurotrophin receptor...

Cellular/Molecular

The p75 Neurotrophin Receptor Can Induce Autophagy andDeath of Cerebellar Purkinje Neurons

Maria L. Florez-McClure,1 Daniel A. Linseman,1 Charleen T. Chu,2 Phil A. Barker,3 Ron J. Bouchard,1 Shoshona S. Le,1

Tracey A. Laessig,1 and Kim A. Heidenreich1

1Department of Pharmacology, University of Colorado Health Sciences Center, and Denver Veterans Affairs Medical Center, Denver, Colorado 80262,2Department of Pathology, Division of Neuropathology, and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School ofMedicine, Pittsburgh, Pennsylvania 15213, and 3Center for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, QuebecH3A 2B4, Canada

The cellular mechanisms underlying Purkinje neuron death in various neurodegenerative disorders of the cerebellum are poorly under-stood. Here we investigate an in vitro model of cerebellar neuronal death. We report that cerebellar Purkinje neurons, deprived of trophicfactors, die by a form of programmed cell death distinct from the apoptotic death of neighboring granule neurons. Purkinje neuron deathwas characterized by excessive autophagic–lysosomal vacuolation. Autophagy and death of Purkinje neurons were inhibited by nervegrowth factor (NGF) and were activated by NGF-neutralizing antibodies. Although treatment with antisense oligonucleotides to the p75neurotrophin receptor (p75ntr) decreased basal survival of cultured cerebellar neurons, p75ntr-antisense decreased autophagy andcompletely inhibited death of Purkinje neurons induced by trophic factor withdrawal. Moreover, adenoviral expression of a p75ntrmutant lacking the ligand-binding domain induced vacuolation and death of Purkinje neurons. These results suggest that p75ntr isrequired for Purkinje neuron survival in the presence of trophic support; however, during trophic factor withdrawal, p75ntr contributesto Purkinje neuron autophagy and death. The autophagic morphology resembles that found in neurodegenerative disorders, suggestinga potential role for this pathway in neurological disease.

Key words: autophagy; Purkinje neuron; p75ntr; cell death; neurotrophin; vacuoles

IntroductionChronic neurodegenerative diseases are characterized by a selec-tive loss of specific neuronal populations over a period of years oreven decades. Although the underlying causes of most neurode-generative diseases are unclear, the loss of neurons and neuronalcontacts is a key feature of disease pathology. Elucidation of thecellular mechanisms regulating neuronal cell death is critical fordeveloping new therapeutic strategies to slow or halt the progres-sive neurodegeneration in these disorders.

Purkinje neurons in the cerebellum integrate input to the cer-ebellar cortex from the mossy fibers and climbing fibers and sub-sequently generate inhibitory output to the deep cerebellar nuclei(Ghez and Thach, 2000). In addition to their essential role inproper cerebellar function, Purkinje neurons provide critical tro-phic support to developing cerebellar granule neurons and infe-rior olivary neurons (Torres-Aleman et al., 1994; Zanjani et al.,

1994; Linseman et al., 2002a). Purkinje neurons are specificallylost in various neurodegenerative conditions such as spinocere-bellar ataxias (Koeppen, 1998; Watase et al., 2002), ataxia telang-ectasia (Gatti and Vinters, 1985; Borghesani et al., 2000), autism(Ritvo et al., 1986; Bailey et al., 1998), and certain prion enceph-alopathies (Ferrer et al., 1991; Watanabe and Duchen, 1993; Las-mezas et al., 1997). Although Purkinje cell loss is a critical featureof disease pathology, the molecular mechanisms underlying Pur-kinje cell death remain poorly understood.

The Lurcher (Lc) mouse has been extensively used as an invivo model of Purkinje neuron degeneration. The cell degenera-tion in the Lurcher cerebellum results from a single point muta-tion in the �2 glutamate receptor (GluR�2), whose expression isrestricted to Purkinje neurons (Zuo et al., 1997). The degenera-tion and loss of Purkinje neurons in Lurcher cerebellum is fol-lowed by a secondary death of cerebellar granule neurons andinferior olivary neurons attributable to loss of trophic supportnormally provided by their afferent target Purkinje neurons(Wetts and Herrup, 1982). Because GluR�2 Lc causes a constitu-tive depolarization of Purkinje neurons, it was originally thoughtthat the death of Purkinje neurons in the Lc cerebellum was akinto excitotoxicity mediated by excessive calcium influx. However,a recent report suggested that the mechanism of GluR�2 Lc-induced Purkinje cell degeneration can be dissociated from de-polarization (Selimi et al., 2003). Instead, Lc Purkinje cell death ishypothesized to involve interactions between the mutant GluR�2

Received Dec. 30, 2003; revised March 16, 2004; accepted March 17, 2004.This work was supported by a Department of Veterans Affairs merit award (K.A.H.), Department of Defense Grant

DAMD17-99-1-9481 (K.A.H.), National Institutes of Health Grants NS38619-01A1 (K.A.H.) and NS40817 (C.T.C.), aDepartment of Veterans Affairs Research Enhancement Program award (K.A.H., D.A.L.), and a National ResearchService award (M.L.F.-M.). We thank Ardith Ries of the University of Pittsburgh electron microscopy laboratory fortechnical assistance and Dr. John Shelburne of the Duke University and Veterans Affairs Medical Centers (Durham,NC) for helpful discussion.

Correspondence should be addressed to Kim A. Heidenreich, Department of Pharmacology, C236, University ofColorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. E-mail: [email protected].

DOI:10.1523/JNEUROSCI.5744-03.2004Copyright © 2004 Society for Neuroscience 0270-6474/04/244498-12$15.00/0

4498 • The Journal of Neuroscience, May 12, 2004 • 24(19):4498 – 4509

receptor and the proteins nPIST and Beclin 1, because these pro-tein–protein interactions can lead to cell death and increase au-tophagy when overexpressed in heterologous cells (Yue et al.,2002). Furthermore, both groups were able to demonstrate theappearance of autophagic vacuoles in Lc Purkinje neurons beforedegeneration, suggesting that upregulated autophagy is an earlyfeature of dying Purkinje neurons (Yue et al., 2002; Selimi et al.,2003).

Autophagy is a degradative pathway responsible for the bulkof proteolysis in normal cells. Autophagy is an evolutionarilyconserved pathway that leads to the degradation of proteins andentire organelles in cells undergoing stress; however, in extremecases it can result in cellular dysfunction and cell death (Klionskyand Emr, 2000). Autophagy begins with the formation of double-membrane vesicles that sequester cytoplasm and organelles inautophagosomes (autophagic vacuoles in mammalian cells). Theautophagosomes fuse with lysosomes, forming autophagolyso-somes. The contents of autophagolysosomes are degraded by ly-sosomal enzymes into basic macromolecules, which are then re-cycled for use in essential cellular functions (Stromhaug andKlionsky, 2001).

Dysregulation of autophagolysosomal activity can lead to celldeath and is implicated in several neurodegenerative conditions,including Parkinson’s disease (Anglade et al., 1997), Alzheimer’sdisease (Cataldo et al., 1996; Nixon et al., 2000), Lewy body de-mentias (Zhu et al., 2003), Huntington’s disease (Kegel et al.,2000; Petersen et al., 2001), and prion encephalopathies (Boel-laard et al., 1991; Liberski et al., 2002). Nutrient deprivation,including withdrawal of serum (Mitchener et al., 1976), is onestimulus known to induce autophagy. In this report, we describean in vitro model to investigate signaling pathways that regulateautophagy and survival in cerebellar Purkinje neurons. We usedcerebellar cultures from early postnatal rats to investigate Pur-kinje neuron death in response to trophic factor withdrawal. Inthis model, Purkinje cell loss was characterized by extensive cy-toplasmic vacuolation and a marked absence of nuclear conden-sation or fragmentation. The vacuoles stained with markers forautophagic vacuoles and lysosomes and the presence of abundant,enlarged autophago(lyso)somes was confirmed by transmissionelectron microscopy. The autophagy inhibitor 3-methyladenine di-minished cytoplasmic vacuolation and moderately increased thesurvival of Purkinje neurons. Nerve growth factor (NGF) also inhib-ited autophagy and death of Purkinje neurons. The protective effectsof NGF were mediated by the low-affinity p75 neurotrophin recep-tor (p75ntr). Our data support the hypothesis that p75ntr can regu-late autophagy and death in Purkinje neurons.

Materials and MethodsMaterials. Polyclonal antibodies to calbindin-D28k and p75ntr, mono-clonal antibodies to NGF, and purified NGF were obtained from Chemi-con (Temecula, CA). Polyclonal antibodies to NGF and monoclonalantibodies to phospho-TrkA were obtained from Santa Cruz Biotechnol-ogy (Santa Cruz, CA). The monoclonal phosphotyrosine antibody (Ab)was obtained from Upstate Cell Signaling Solutions (Lake Placid, NY). Apolyclonal antibody to the intracellular domain of rat p75ntr wasobtained from Covance (Berkeley, CA). Cy3- and FITC-conjugatedsecondary antibodies for immunocytochemistry were purchased fromJackson ImmunoResearch (West Grove, PA). Horseradish peroxidase-linked secondary antibodies and reagents for enhanced chemiluminescencedetection were obtained from Amersham Biosciences (Piscataway, NJ). Ly-sosensor blue was obtained from Molecular Probes (Eugene, OR). Mono-dansylcadaverine, 3-methyladenine, and 4,6-diamidino-2-phenylindole(DAPI) were from Sigma (St. Louis, MO). Adenoviral cytomegalovirus(CMV; negative control adenovirus) was from Dr. Jerry Schaack (University

of Colorado Health Sciences Center, Denver, CO). The adenoviral ratp75ntr myristylated intracellular domain (p75mICD) has been describedpreviously (Roux et al., 2001).

Cell culture. Rat cerebellar granule neurons were isolated from 7-d-oldSprague Dawley rat pups as described previously (D’Mello et al., 1993).Briefly, neurons were plated at a density of 2.0 � 10 6 cells/ml in basalmodified Eagle’s medium (BME) containing 10% fetal bovine serum, 25mM KCl, 2 mM L-glutamine, and penicillin (100 U/ml)-streptomycin (100�g/ml; Invitrogen, Gaithersburg, MD) Cytosine arabinoside (10 �M) wasadded to the culture medium 24 hr after plating to limit the growth ofnon-neuronal cells. Experiments were performed after 7 d in culture. Deathwas induced by removing the serum- and high-potassium-containing mediaand replacing it with serum-free and 5 mM potassium BME.

Adenoviral infection. Five days after plating, neuronal cultures wereinfected with either control adenovirus (adenoviral CMV) or adenoviralp75ntr, full-length or truncated, mICD, each at the indicated multiplicityof infection. After infection, cells were returned to the incubator for 48 hrat 37°C and 10% CO2. On day 7, neurons were processed for live cellimaging or fixed for immunocytochemistry.

Antisense. The p75ntr antisense and missense oligonucleotides (bothat 5 �M) were added to the cultures at the time of plating [day in vitro(DIV) 0] by repeatedly triturating the cells in the presence of the oligo-nucleotides before seeding. The oligonucleotides were present through-out the culture. Trophic factor withdrawal was performed on day 5 or 6in culture (DIV 5 or 6) for either 24 or 48 hr. After this treatment, the cellswere processed for live cell lysosensor experiments or fixed for immuno-cytochemistry to count Purkinje neurons. In all cases, the antisense wastaken up by neurons with nearly 100% efficiency as assayed by visualiza-tion of the fluorescently labeled oligonucleotides. HPLC-purified phos-phorothioate oligonucleotides were purchased from Integrated DNATechnologies (Coralville, IA). Sequences used were as follows: rat p75ntr5� antisense, 5�-ACCTGCCCTCCTCATTGCA-3�; and rat p75ntr 5�missense, 5�-CTCCCACTCGTCATTCGAC-3�. The rat 5� p75ntr anti-sense was also purchased with a 5� 56-FAM fluorescent label so thatuptake by the neurons could be monitored. The antisense sequence usedhas been characterized previously and has been shown to be effective atinhibiting p75ntr-mediated cell death both in vitro and in vivo (Barrettand Bartlett, 1994; Cheema et al., 1996; Lowry et al., 2001).

Immunocytochemistry. Neuronal cultures were plated on polyethyl-eneimine-coated glass coverslips. Neurons were infected and induced toundergo death as indicated previously. After treatment, the neurons werefixed with 4% paraformaldehyde and then permeabilized and blockedwith PBS, pH 7.4, containing 0.2% Triton X-100 and 5% BSA. Cells werethen incubated with polyclonal antibodies against calbindin (1:250), ac-tive caspase-3 (1:500), or p75ntr (1:250) overnight at 4°C diluted in PBScontaining 0.1% Triton X-100 and 2% BSA. Primary antibodies werethen removed, and the cells were washed at least six times with PBS atroom temperature. The neurons were then incubated with Cy3-conjugated donkey anti-rabbit secondary antibodies (1:500) and DAPI(1 �g/ml) for 1 hr at room temperature. The cells were then washed atleast six more times with PBS, and coverslips were adhered to glass slideswith mounting medium (0.1% p-phenylenediamine in 75% glycerol inPBS). Imaging was performed on a Zeiss (Thornwood, NY) Axioplan 2microscope equipped with a Cooke Sensicam deep-cooled CCD camera,and images were analyzed with the Slidebook software program (Intelli-gent Imaging Innovations Inc., Denver, CO).

Purkinje cell counts. Purkinje neuron numbers were measured bycounting the number of calbindin-positive cells in 152 fields under 63�oil that were randomly selected by following a fixed grid pattern over thecoverslip. The total area counted per coverslip was 14.6 mm 2 or �13% ofthe coverslip. In addition to calbindin staining, cells were also judged ontheir morphology. Cells counted as Purkinje neurons had large roundedcell bodies, elaborate processes, and larger, more oval nuclei (in compar-ison with the smaller, rounder nuclei of granule neurons). Some verybright calbindin-positive cells that were either as small as the granuleneurons, having bipolar processes, or very large flattened cells, lackingelaborate processes, were not counted because previous studies on invitro differentiation of Purkinje neurons suggest that these cells mayrepresent examples of delayed or aberrant development of Purkinje neu-

Florez-McClure et al. • Purkinje Autophagic Death Induced by p75ntr J. Neurosci., May 12, 2004 • 24(19):4498 – 4509 • 4499

rons in culture (Baptista et al., 1994). At least threecoverslips were counted per experimental condi-tion. The numbers were averaged and expressedas a percentage of the average number counted inthe appropriate controls. This was repeated for atleast three independent experiments.

Live cell imaging and vacuolation measure-ments. Lysosensor blue and monodansylcadav-erine (MDC) were added to the cultures at theend of the indicated treatments and returned tothe incubator for 20 min. The cells were thenwashed three times with 37°C phenol red-freeDMEM to remove nonspecifically bound dyes.The coverslips were mounted onto glass slideson �10 �l of phenol red-free DMEM; excessmedia were aspirated; and the coverslips weresealed and imaged immediately. Purkinje neu-rons were identified on the basis of their mor-phology (large, oval nuclei, large cell body, andmultiple processes) by scanning the coverslipsunder bright-field differential interference con-trast with a 63� oil objective. Images of thelysosensor blue and MDC fluorescence werecaptured on the DAPI and FITC channels, re-spectively, with the 100� oil objective. The lyso-sensor blue images were used to measure the di-ameters of all the visible lysosomes in at least sixPurkinje neurons per condition. This was usuallybetween 100 and 200 lysosomes. This was re-peated for at least three independent experiments.

Transmission electron microscopy. Culturedcells were fixed in 2% paraformaldehyde and2.5% glutaraldehyde for 1 hr, postfixed in 1% os-mium tetroxide for 1 hr at 4°C, and processed forembedding in the culture dish. Cells were thengently scraped and embedded in blocks of Epon-araldite. Thin sections were stained with 4% aque-ous uranyl acetate and lead citrate and examinedon a Philips CM-12 electron microscope.

Lysate preparation. After incubation for theindicated times and with the reagents specifiedabove, the culture medium was aspirated, cellswere washed once with 2 ml of ice-cold PBS(PBS, pH 7.4), placed on ice, and scraped intolysis buffer (200 �l/35 mm well) containing 20mM HEPES, pH 7.4, 1% Triton X-100, 50 mM

NaCl, 1 mM EGTA, 5 mM �-glycerophosphate,30 mM sodium pyrophosphate, 100 �M sodiumorthovanadate, 1 mM phenylmethylsulfonylfluoride, 10 �g/ml leupeptin, and 10 �g/mlaprotinin. Cell debris was removed by centrifu-gation at 6000 � g for 3 min and the proteinconcentration of the supernatant was deter-mined using a commercially available proteinassay kit (Pierce, Rockford, IL). Equal amountsof supernatant protein were diluted to a finalconcentration of 1� SDS-PAGE sample buffer,boiled for 5 min, and electrophoresed through7.5% polyacrylamide gels. Proteins were trans-ferred to polyvinylidene membranes (Milli-pore, Bedford, MA) and processed for immu-noblot analysis.

Immunoblot analysis. Nonspecific bindingsites were blocked in PBS, pH 7.4, containing0.1% Tween 20 (PBS-T) and 1% BSA for 1 hr atroom temperature. Primary antibodies were diluted in blocking solutionand incubated with the membranes for 1 hr. Excess primary Ab wasremoved by washing the membranes three times in PBS-T. The blotswere then incubated with the appropriate horseradish peroxidase-

conjugated secondary Ab diluted in PBS-T for 1 hr and were subse-quently washed three times in PBS-T. Immunoreactive proteins weredetected by enhanced chemiluminescence. In some experiments, mem-branes were reprobed after stripping in 0.1 M Tris-HCl, pH 8.0, 2% SDS, and

Figure 1. Trophic factor withdrawal induces a nonapoptotic death pathway in Purkinje neurons. Cultured cerebellar neuronswere allowed to differentiate in medium containing 10% fetal calf serum and 25 mM potassium. On day 7, neurons were eithermaintained in control medium (25K�Ser) or subjected to trophic factor withdrawal medium (5K�Ser) for 24 – 48 hr. A, Purkinjenumber was quantified by counting the total number of calbindin-positive Purkinje cells in randomly chosen, equally sized areasper condition (total area, 14.6 mm 2). Numbers are plotted as a percentage of control. Values represent the mean � SEM of threeindependent experiments each performed in triplicate. **Significant difference from 25K�S control at p � 0.01, one-wayANOVA, Tukey’s post hoc test. B–G, Cells incubated in either control medium ( B–D) or deprived of trophic factors ( E–G) were fixedand stained with polyclonal antibodies against calbindin-D28k (a specific marker of Purkinje neurons; red) and the nuclear dyeDAPI (blue). B, C, Purkinje neurons maintained in control medium demonstrate differentiated morphology. D, DAPI stainingreveals the nuclei of healthy granule neurons and a Purkinje neuron (arrow). E, F, Trophic factor withdrawal induced extensivecytoplasmic vacuolation of Purkinje neurons (see magnified insets). G, DAPI staining reveals increased nuclear condensation andfragmentation in granule neurons, whereas there is a notable lack of nuclear condensation in Purkinje nuclei (arrow).

4500 • J. Neurosci., May 12, 2004 • 24(19):4498 – 4509 Florez-McClure et al. • Purkinje Autophagic Death Induced by p75ntr

100 mM �-mercaptoethanol for 30 min at 52°C. The blots were rinsed twicein PBS-T and processed as above with a different primary Ab. Autolumino-grams shown are representative of at least three independent experiments.

Data analysis. Results shown represent the means � SEM for the num-ber of independent experiments performed. Statistical differences be-tween the means of unpaired sets of data were evaluated using one-wayANOVA followed by post hoc Tukey’s test; p � 0.05 was consideredstatistically significant.

ResultsTrophic factor withdrawal results in a loss of Purkinjeneurons that is morphologically distinct from apoptosisWe have investigated Purkinje neuron death induced by trophicfactor withdrawal using primary cerebellar neuronal cultures. Al-though these cultures have been extensively used to study signal-ing pathways that regulate survival of cerebellar granule neurons(D’Mello et al., 1993; Linseman et al., 2002b; Vaudry et al., 2003),they also provide a model system for studying differentiated Pur-kinje neurons (Baptista et al., 1994). Primary neuronal cultures

are derived from dissociated cerebella of early postnatal rats.These neuronal cultures survive and differentiate when main-tained in medium containing 10% fetal calf serum and a depo-larizing concentration of potassium (25 mM). Granule neurons,which constitute �98% of the cell population, undergo classicalapoptosis, characterized by chromatin condensation and caspaseactivation, when they are deprived of serum and depolarizingpotassium (trophic factor withdrawal; (D’Mello et al., 1993; Lin-seman et al., 2002b).

In contrast, Purkinje neurons, which make up �2% of the

Figure 2. Autophagic and lysosomal markers stain vacuoles that form during Purkinje neu-ron degeneration. Purkinje neurons maintained in either control (25K�Ser) or trophic factorwithdrawal (5K�Ser) media for 24 hr were stained with the autophagic marker MDC (green),and lysosensor blue, a pH-sensitive dye that fluoresces blue in acidic environments. Live cellimaging of control ( A–D) and trophic factor-deprived ( E–H) Purkinje neurons was performed.A, E, Bright-field images of cells identified as Purkinje neurons based on their morphology,characterized by a large cytoplasm (compared with granule neurons) and extensive neuronalprocesses. B, F, MDC staining demonstrating increases in autophagosomes in Purkinje neuronsafter trophic factor withdrawal. C, G, Lysosensor blue staining reveals acidic organelles (i.e.,lysosomes) and demonstrates a marked increase in the size of lysosomes in Purkinje neuronsafter withdrawal of trophic factors. D, H, All three fields merged, demonstrating colabeling ofsome cytoplasmic vacuoles with both MDC and lysosensor blue indicative of autophagolysoso-mal fusion.

Figure 3. The autophagy inhibitor 3MA, blocks the increased vacuolation and loss of Pur-kinje neurons. Purkinje neurons were maintained in either control medium (25K�S) or trophicfactor withdrawal medium (5K�S) in the absence or presence of 5 mM 3MA (5K�S�3MA) for24 hr. A, The effects of the various treatments on vacuole size were quantified by measuring thediameters of all visible lysosensor blue-positive vacuoles in 6 –12 Purkinje neurons per treat-ment condition in at least three independent experiments. Usually, the total number of vacu-oles measured per condition was between 100 and 200. The size distribution was graphed as apercentage of total vacuoles that were within the indicated size ranges. B, Quantitation of theeffects of 3MA on Purkinje neuron numbers demonstrate that addition of 5 mM 3MA to trophicwithdrawal medium partially rescues Purkinje neurons from death after 24 hr of trophic factorwithdrawal, compared with healthy controls. Numbers are mean � SEM values of seven inde-pendent experiments, each performed in triplicate. **,*Significant difference from 25K�S(**p � 0.01; *p � 0.05). ‡‡,‡Significant difference from 5K�S (‡‡p � 0.01; ‡p � 0.05).


culture, undergo a distinct nonapoptoticform of cell death in response to trophicfactor withdrawal. Quantification of thenumber of Purkinje neurons after trophicfactor withdrawal is shown in Figure 1A.Purkinje neurons were identified by stain-ing with antibodies to the Purkinje markercalbindin-D28k. After 24 or 48 hr of tro-phic factor withdrawal, Purkinje numberswere 50.4 � 1.4 and 22.3 � 7.0% of con-trols, respectively. The remaining Purkinjeneurons showed markedly different mor-phology from that of control cells, charac-terized by extensive cytoplasmic vacuola-tion (Fig. 1, compare control cells, B, C,with trophic factor-deprived cells, E, F). Incontrast to granule neurons deprived oftrophic support, which demonstrated sub-stantial nuclear condensation and frag-mentation characteristic of apoptosis (Fig.1, compare D, G), Purkinje neuronsshowed no obvious signs of nuclear con-densation or fragmentation (Fig. 1, com-pare nuclei indicated in D, G, arrows). Thedegenerating Purkinje neurons did notstain with propidium iodide, indicatingthat they retained membrane integritythroughout the death process (data notshown). The maintenance of membraneintegrity suggested that Purkinje death didnot occur by necrosis. Vacuole formationand subsequent loss of Purkinje neuronsrequired new RNA synthesis because addi-tion of the transcriptional inhibitor acti-nomycin D (ActD) decreased vacuolationand significantly increased the numbers ofPurkinje cells to 78.7 � 1.5% comparedwith 50.4 � 1.4% ( p � 0.05) after 24 hr oftrophic factor withdrawal. The ability ofActD to protect Purkinje neurons furthersuggested that the mechanism of death wasnot necrotic but was rather a regulated program of cell suicide.

Purkinje neurodegeneration is associated withincreased autophagyExtensive cytoplasmic vacuolation was the most prominent mor-phological feature distinguishing healthy from degenerating Pur-kinje neurons in response to trophic factor withdrawal. To deter-mine the mechanism underlying the loss of Purkinje neurons, wefirst characterized the nature of the vacuoles formed during with-drawal conditions. The formation of extensive cytoplasmic vacu-oles is consistent with the upregulation of autophagy. Theautofluorescent drug MDC has been shown to specifically accu-mulate in autophagic vacuoles (Biederbick et al., 1995; Munafoand Colombo, 2001). To determine whether autophagy was ac-tivated in Purkinje neurons subjected to trophic factor with-drawal, we incubated the cerebellar cultures with MDC and thenvisualized the MDC staining using live cell imaging techniques.In addition to MDC, the neurons were stained with lysosensorblue, a pH-sensitive dye that fluoresces in acidic compartments(pKa, 5.2), thus specifically revealing lysosomes. These experi-ments demonstrated that control Purkinje neurons containedboth lysosomal and autophagic vacuoles. However, in healthy

Purkinje neurons these vacuoles were on average very small (Fig.2A–D). In contrast, 24 hr of trophic factor withdrawal induced amarked increase in the sizes of autophagic and lysosomal vacu-oles, as detected by MDC and lysosensor blue staining, respec-tively (Fig. 2E–H).

To determine whether augmented autophagy contributed toPurkinje neuron death, we incubated the cultures with 5 mM

3-methyladenine (3MA), a drug known to inhibit the formation ofautophagosomes (Seglen and Gordon, 1982). Quantitative analysisrevealed that trophic factor withdrawal significantly increased thesize of vacuoles in Purkinje neurons (Fig. 3A). In healthy Purkinjeneurons, �89% of the lysosensor blue-positive vacuoles were �0.75�m in diameter. After 24 hr of trophic factor withdrawal, most(�47%) of the lysosensor blue-positive vacuoles in the dying Pur-kinje neurons were much larger with a diameter, between 0.75 and1.5 �m, and a significant percentage (�32%) were �1.5 �m indiameter. Addition of 5 mM 3MA to the cerebellar cultures almostcompletely maintained the lysosensor size profile of vacuoles in therange observed in control Purkinje neurons. The inhibition of auto-phagic activity by 3-methyladenine correlated with significantly in-creased numbers of Purkinje neurons that remained after 24 hr oftrophic factor deprivation (Fig. 3B).

Figure 4. Transmission electron microscopy of Purkinje neurons after 24 hr of trophic factor withdrawal demonstrates ultra-structural features of autophagy. A, Low-power micrograph of a large Purkinje cell with multiple secondary lysosomes (whitearrows) and a large autophagic vacuole (asterisk). In contrast, the smaller cerebellar granule neuron (black arrow) does not showthese changes. B, Detail of the large autophagic vacuole indicated by the asterisk. There are disorganized membranous structuresconsistent with endoplasmic reticulum, ribosomes, and glycogen, delimited predominantly by a single membrane (white arrow-heads). C, Micrograph showing an autophagosome delimited by a double membrane (white arrows). Note the similarity of thecontents to adjacent mitochondria and cytoplasmic glycogen (lower left). Scale bars, 500 nm.


Electron microscopic analysis of autophagic vacuoles introphic factor-deprived Purkinje neuronsTaken together, the above data suggested that autophagy is up-regulated in Purkinje neurons subjected to trophic factor with-drawal. Electron microscopy was performed on cerebellar cul-tures to confirm that the enlarged MDC- and lysosensor-positivevacuoles observed after trophic factor withdrawal were indeedautophagic vacuoles. The electron micrographs revealed that thecytoplasm of identified Purkinje cells showed multiple autopha-gosomes, multivesicular bodies, and other secondary lysosomesranging in size from 0.7 to 3.0 �m after 24 hr of trophic factorwithdrawal (Fig. 4). Some of these autophagic structures con-tained ribosomes, glycogen, and disorganized membranousorganelles (Fig. 4A,B, asterisks, C). Occasionally, a double mem-brane was observed extending around cytoplasmic contents (Fig.4C, white arrows), indicative of earlier stages of autophagy (Shel-burne et al., 1973). The cytoplasm of Purkinje neurons also con-tained lipid droplets (Fig. 4A). In Purkinje neurons, the nuclearchromatin was uniformly dispersed (Fig. 4A). In contrast to thePurkinje neurons, large autophagolysosomes were absent fromthe granule neurons, which instead showed mild cytoplasmiccondensation and peripheral chromatin clumping (Fig. 4A, blackarrow).

Purkinje neuron autophagy and deathare caspase-independentPrevious studies have shown that trophic factor withdrawal andother death-inducing stimuli can simultaneously induce both au-tophagy and apoptosis (Jia et al., 1997; Xue et al., 1999; Uch-iyama, 2001). In these models, caspase activation and apoptosisoccurred downstream of autophagy and could be blocked by3MA. To determine whether the autophagic death pathway inPurkinje neurons acted in concert with caspase activation, weadded the broad-spectrum caspase inhibitor zVAD-FMK to Pur-kinje neurons undergoing trophic factor withdrawal. Addition ofzVAD-FMK (100 �M), which effectively inhibits caspase activityin cerebellar granule neurons, was unable to block Purkinje vac-uolation or death. The numbers of Purkinje neurons remainingafter 24 hr of trophic factor withdrawal in the absence and pres-ence of zVAD-FMK were 55.7 � 2.8 and 54.7 � 3.5, respectively.Furthermore, we were unable to detect increases in activatedcaspase-3 by immunocytochemical techniques in Purkinje neu-rons undergoing death induced by trophic factor withdrawal(data not shown). In contrast, activated caspase-3 immunoreac-tivity increases markedly in the granule neurons after trophicfactor withdrawal (Linseman et al., 2003). These results suggestthat neither autophagy nor death of Purkinje neurons requiredcaspase activation.

NGF promotes survival and decreases autophagy inPurkinje neuronsHaving established that trophic factor withdrawal induced auto-phagy and death of cerebellar Purkinje neurons, we next exam-ined whether neurotrophin signaling could protect Purkinje neu-rons in this model. NGF is a neurotrophin known to promote thesurvival and differentiation of many types of neurons both invitro and in vivo, including Purkinje neurons (Legrand and Clos,1991; Cohen-Cory et al., 1993; Mount et al., 1998). We addedNGF at various concentrations to the cultures at the time of tro-phic factor withdrawal and determined its effect on Purkinje sur-vival. High concentrations of NGF (�50 ng/ml) significantly in-creased Purkinje numbers compared with trophic factorwithdrawal alone (Fig. 5E). To determine whether NGF could

prevent extensive autophagic vacuolation, cultures were sub-jected to trophic factor withdrawal in the absence or presence ofNGF, and quantitative analysis of vacuolation was performedusing live cell lysosensor measurements (Fig. 5G). Again, in thepresence of trophic support, the majority of Purkinje neuronscontained small (�0.75 �m) lysosensor-positive vacuoles. After24 hr of trophic factor withdrawal, most Purkinje neurons con-tained very large vacuoles, with 49% of vacuoles between 0.75 and1.5 �m, �21% of vacuoles between 1.5 and 2.25 �m, and �13%of vacuoles �2.25 �m. The addition of NGF (2.5 �g/ml) to thetrophic factor withdrawal media significantly decreased the over-all vacuolation of Purkinje neurons. The lysosome size profilewas shifted, with the majority of vacuoles (�76%) being �0.75�m in diameter, �19% of vacuoles between 0.75 and 1.5 �m,�4% of vacuoles between 1.5 and 2.25 �m, and �1% of vacuoles�2.25 �m. Images shown are representative of the overall effectsof the various treatments (Fig. 5A–C).

To determine whether decreasing NGF in the cerebellar cul-tures would be sufficient to induce autophagy and death of Pur-kinje neurons, we added NGF-neutralizing antibodies to the me-dia and determined the effects on Purkinje survival andvacuolation. Addition of NGF-neutralizing antibodies to the cul-tures for 24 hr reduced the numbers of Purkinje neurons to70.6 � 5.3% of controls (Fig. 5F). Control IgG had no effect onthe number of Purkinje neurons. The effect of the NGF-neutralizing antibodies was less than the effect of complete tro-phic factor withdrawal, which reduced Purkinje numbers to45.2 � 2.7% of control (Fig. 5F). To determine whether theNGF-neutralizing antibodies could induce autophagic vacuolationin Purkinje neurons, we performed live cell imaging experiments tomeasure the sizes of Purkinje lysosomes (Fig. 5H). Again, controlPurkinje neurons contained mostly small lysosensor-positive vacu-oles, whereas 24 hr of trophic factor withdrawal markedly inducedthe appearance of much larger vacuoles. Addition of NGF-neutralizing antibodies similarly increased the appearance oflarger vacuoles in Purkinje neurons, although not to the sameextent as trophic factor withdrawal. Control IgG had no effect onthe size distribution of the vacuoles. The ability of NGF-neutralizing antibodies to induce the formation of vacuoles wasalso observed by calbindin staining (Fig. 5, compare A, D).

The protective effects of NGF on Purkinje neurons areindependent of TrkA signalingNGF exerts its effects by binding to two distinct receptors, theTrkA receptor and p75ntr. The high concentrations of NGF re-quired to promote Purkinje survival suggested that the p75ntrreceptor mediated the neuroprotective effects of NGF in thesecultures. Further data supporting this hypothesis come from ex-periments showing that TrkA receptor tyrosine phosphorylationis high under basal conditions and does not change on trophicfactor withdrawal (up to 6 hr) or on addition of high doses ofNGF (Fig. 6A,B). Furthermore, Purkinje neurons demonstratedsignificantly increased autophagic vacuolation (within 3– 4 hr oftrophic factor withdrawal, as assessed by live cell lysosensor mea-surements of vacuole size; Fig. 6C) before any significant decreasein TrkA activation.

In the presence of trophic factors, the p75ntr supportsPurkinje neuron survivalThe p75 neurotrophin receptor is expressed throughout the de-veloping cerebellum and demonstrates specific spatial and tem-poral regulation (Yan and Johnson, 1988; Carter et al., 2003). In


particular, p75ntr is expressed in the de-veloping Purkinje neurons by postnatalday 7, the time point at which we obtainedour cerebellar cultures. p75ntr immunore-activity gradually decreases until it be-comes absent from adult cerebellum; how-ever, injury such as axotomy of Purkinjeneurons results in marked re-expression ofp75ntr in the injured neurons (Martinez-Murillo et al., 1993). These data suggest thatp75ntr is involved in both the develop-ment of the cerebellum and injury re-sponses in Purkinje neurons.

To determine whether p75ntr was in-volved in mediating autophagy and deathof Purkinje neurons, cerebellar cultureswere incubated with antisense oligonucle-otides directed against a 5� region span-ning the initiation codon of the rat p75ntrmRNA. This antisense sequence has beenshown to be effective at preventing theNGF withdrawal or axotomy-induceddeath of dorsal root ganglion neurons(Barrett and Bartlett, 1994; Cheema et al.,1996) and the axotomy-induced death ofspinal motor neurons (Lowry et al., 2001),which are mediated by p75ntr. Dissociatedcerebellar tissue was triturated with 5 �M

56-FAM-labeled antisense or missensephosphorothioate oligonucleotides at thetime of plating. The antisense entered bothgranule neurons and Purkinje neurons inthe culture, with nearly 100% efficiency asassessed by fluorescence microscopy. Thep75ntr antisense had two distinct effectson the neurons in the culture. Inclusion ofthe antisense in the culture medium for 7 dresulted in a significant decrease in thebasal survival of both cerebellar granuleand Purkinje neurons. Purkinje neuronnumber with the p75ntr antisense was35 � 2.3% of control cultures. The effectof the p75ntr antisense on cell numberswas specific because cultures treated withscrambled missense oligonucleotides didnot demonstrate diminished survival.Also, the loss of cells induced by p75ntrantisense was not apparent until approxi-mately day 4 or 5 in culture (as assed by celldensity by bright-field examination of thecultures). This corresponds to the time inculture when NGF production is first detect-able by Western blot analysis (data notshown). These findings suggest that p75ntrmediates survival in response to autocrineand paracrine neurotrophic factors suchas NGF and possibly other neurotrophinssuch as brain-derived neurotrophic factor(BDNF) or neurotrophin-3 (NT-3), whichare known to be secreted by cells in thesecerebellar cultures (Favaron et al., 1993; Le-ingartner et al., 1994; Condorelli et al., 1998;Marini et al., 1998; Bhave et al., 1999).

Figure 5. The neurotrophin NGF blocks the loss and the increased vacuolation of Purkinje neurons. A–D, Purkinje neuronsmaintained in various conditions were fixed and stained with antibodies to calbindin-D28k (a specific marker of Purkinje neurons;red) and the nuclear dye DAPI (blue). Images shown are representative of the effects of the various treatments. Purkinje neuronswere maintained in control medium (A, 25K�S), trophic factor withdrawal medium (B, 5K�S), trophic factor withdrawalmedium with 2500 ng/ml NGF (C, 5K�S�NGF), and control medium with 2 �g/ml NGF-neutralizing antibodies (D,25K�S��NGF) for 24 hr. E, Quantitation of the effects of NGF on Purkinje neuron numbers demonstrates that NGF partiallyrescues Purkinje neurons from death in a concentration-dependent manner after 24 hr of trophic factor withdrawal, comparedwith healthy controls. F, Quantitation of the effects of NGF-neutralizing antibodies on Purkinje neuron numbers demonstratesthat depleting NGF from control media results in a loss of Purkinje neurons, whereas control antibodies had no effect on Purkinjesurvival. Numbers are mean � SEM values of at least three independent experiments, each performed in triplicate. G, H, The


p75ntr promotes autophagy and death of Purkinje neurons inthe absence of trophic stimuliIn contrast to its effects on basal survival, the p75ntr antisensecompletely blocked the death of Purkinje neurons induced by 48hr of trophic factor withdrawal (Fig. 7A). Moreover, 5 �M anti-sense decreased the autophagic vacuolation of Purkinje neuronselicited by 24 hr of trophic factor withdrawal (Fig. 7B). Again, inthe presence of trophic support, most (�90%) of the Purkinjeneuron lysosensor-positive vacuoles were �0.75 �m, and only�10% of vacuoles were between 0.75 and 1.5 �m, with no vacu-

oles �1.5 �m in diameter After 24 hr of trophic factor with-drawal, most Purkinje neurons contained very large vacuoles,with �49% of vacuoles between 0.75 and 1.5 �m, �13% of vacu-oles between 1.5 and 2.25 �m, and �11% of vacuoles �2.25 �m.Cultures that were preincubated with p75ntr antisense oligonu-cleotides demonstrated a shifted lysosome size profile, with�70% of vacuoles �0.75 �m in diameter, �26% of vacuolesbetween 0.75 and 1.5 �m, �4% of vacuoles between 1.5 and 2.25�m, and �1% of vacuoles �2.25 �m. In contrast, missense oli-gonucleotides had no effect on the lysosome size profile of Pur-kinje neurons deprived of trophic support (Fig. 7B). Collectively,the above results indicate that antisense-mediated depletion ofp75ntr decreases the basal survival of Purkinje neurons in thepresence of trophic support. However, under conditions of tro-phic factor withdrawal, decreasing p75ntr provides a survival ad-vantage to the remaining Purkinje neurons. The ability of p75ntrto mediate both the survival and death in the same neuron de-pending on cellular context is consistent with previous reports ofits effects on dorsal root ganglion neuron survival (Barrett andBartlett, 1994; Sorensen et al., 2003).

Overexpression of a truncated p75ntr lacking theextracellular domain induces Purkinje neuron autophagy anddeath in the presence of trophic supportTo provide direct evidence that the p75ntr can induce autophagyand death in Purkinje neurons, we investigated the effects ofadenoviral-mediated overexpression of a myristoylated ratp75ntr intracellular domain (p75mICD) protein. This p75ntrtruncation mutant was used because it lacks the ligand-bindingdomain, which would complicate the interpretation of resultsbecause of the presence of endogenous neurotrophins in thesecultures. It has been shown that relatively low expression levels ofthis p75ntr mutant mediate the survival of PC12 cells. In contrast,high levels of p75mICD expression induce efficient PC12 celldeath (Roux et al., 2001). Finally, it has been demonstrated thatthe intracellular domain possesses the potent cell death-inducingactivity of p75ntr (Majdan et al., 1997; Coulson et al., 2000; Rabi-zadeh et al., 2000; Murray et al., 2003). Cultures were infectedwith recombinant control CMV or p75mICD adenoviruses atvarious multiplicities of infection (moi) on day 5 in culture. Afterincubation for 48 hr, the cultures were fixed and stained withpolyclonal antibodies raised against the intracellular domain ofrat p75ntr (Majdan et al., 1997), followed by a Cy3-conjugatedsecondary antibody. Increases in p75ntr immunoreactivity afterinfection with the p75mICD adenovirus were primarily and con-sistently observed in cells with morphologies typical of Purkinjeneurons. Semiquantitative analysis of fluorescence images indi-cated that p75ntr expression in p75mICD-infected Purkinje neu-rons was increased by 3.4-fold compared with uninfected con-trols or cells infected with control CMV adenovirus. Todetermine whether overexpression of p75mICD could inducePurkinje neuron death, infected neurons were fixed and stainedwith calbindin antibodies to quantify Purkinje numbers. Overex-pression of p75mICD resulted in a dose-dependent loss of Pur-

kinje neurons, with infection at 50 moiyielding similar levels of Purkinje cell loss,as was observed with trophic factor with-drawal (Fig. 8A). Infection with a controladenovirus at 50 moi did not induce sig-nificant Purkinje neuron death (Fig. 8A).To determine whether overexpression ofp75mICD could induce autophagic vacu-olation in Purkinje neurons, live cell quan-

Figure 6. The protective effects of NGF are not mediated by TrkA. Immunoblot analysis wasperformed to determine the effects of trophic factor withdrawal and various doses of NGF onTrkA activation. A, Immunoblot of samples from cultures subjected to 0, 1, 2, 4, and 6 hr oftrophic factor withdrawal with a phosphotyrosine-specific Ab. B, Immunoblot of samples fromcultures subjected to acid wash, followed by 2 hr of trophic factor withdrawal and then incuba-tion with 0, 5, 50, 500, or 1000 ng/ml of NGF for 10 min. C, The effects of a short time course oftrophic factor withdrawal on vacuole size were quantified by measuring the diameters of allvisible lysosensor blue-positive vacuoles in 6 –12 Purkinje neurons per treatment condition inat least three independent experiments. Usually, the total number of vacuoles measured percondition was between 100 and 200. The size distribution was graphed as a percentage of totalvacuoles that were within the indicated size ranges. **,*Significant difference from 25K�S(**p � 0.01; *p � 0.05).

4

effects of the various treatments on vacuole size were quantified by measuring the diameters of all visible lysosensor blue-positivevacuoles in 6 –12 Purkinje neurons per treatment condition in at least three independent experiments. Usually, the total numberof vacuoles measured per condition was between 100 and 200. The size distribution was graphed as a percentage of total vacuolesthat were within the indicated size ranges. G, A high concentration of NGF (2500 ng/ml) significantly decreased the autophagicvacuolation of Purkinje neurons induced by 24 hr of trophic factor withdrawal. H, NGF-neutralizing antibodies significantlyincreased the vacuolation of Purkinje neurons in control media. E–H, **,*Significant difference from 25K�S (**p � 0.01; *p �0.05). ‡‡,‡Significant difference from 5K�S (‡‡p � 0.01; ‡p � 0.05).


tification of lysosome size was performed (Fig. 8B). Purkinjeneurons that were infected with 50 moi of p75mICD adenovirusdemonstrated significantly increased autophagic vacuolation,with only �58% of vacuoles �0.75 �m, �32% of vacuoles be-tween 0.75 and 1.5 �m, �7% of vacuoles between 1.5 and 2.25�m, and �4% of vacuoles �2.25 �m. Infection with 50 moi ofcontrol CMV adenovirus did not significantly increase Purkinjeneuron vacuolation. In addition to increasing autophagic vacuo-lation, p75mICD infection caused notable membrane blebbingand, in some rare cases, nuclear condensation and fragmentation

in Purkinje neurons. Our observations of increased autophagywith p75mICD expression are consistent with a previous reportof p75ntr intracellular domain-induced death being morpholog-ically characterized by increased vacuolation (Coulson et al.,2000), in addition to the regularly reported morphological fea-tures of apoptosis.

DiscussionMorphological studies of diseased human postmortem brainconsistently reveal signs of increased autophagic activity in de-generating neuronal populations. There are multiple reports inthe literature that describe autophagic vacuoles, as well as distur-bances in the lysosomal degradative system in neurodegenerativeconditions such as Alzheimer’s disease (Cataldo et al., 1996;Nixon et al., 2000), Parkinson’s disease (Anglade et al., 1997),

Figure 7. p75ntr antisense inhibits the loss and vacuolation of Purkinje neurons. Purkinjeneurons maintained in various conditions were fixed and stained with antibodies to calbindin-D28k. A, Quantitation of the effects p75ntr antisense (AS) and missense (MS) oligonucleotides(oligo) on Purkinje neuron numbers expressed as the percentage of the appropriate controldemonstrates that p75ntr antisense completely blocks the loss of Purkinje neurons induced bytrophic factor withdrawal, whereas missense oligos had no effect on Purkinje survival. Numbersare mean � SEM values of at least three independent experiments, each performed in tripli-cate. B, The effects of the various treatments on vacuole size were quantified by measuring thediameters of all visible lysosensor blue-positive vacuoles in 6 –12 Purkinje neurons per treat-ment condition in at least three independent experiments. Usually, the total number of vacuolesmeasured per condition was between 100 and 200. The size distribution was graphed as a percentageof total vacuoles that were within the indicated size ranges. **,*Significant difference from 25K�S(**p � 0.01; *p � 0.05). ‡‡,‡Significant difference from 5K�S (‡‡p � 0.01; ‡p � 0.05).

Figure 8. Overexpression of a truncated p75ntr receptor lacking the ligand-binding domaininduces the loss and vacuolation of Purkinje neurons. Cultures were subjected to trophic factorwithdrawal or adenoviral infection with either control CMV (at 50 moi) or myristoylated intra-cellular domain p75ntr (at both 10 and 50 moi) adenoviruses on DIV 5. A, The cells were fixedand stained with calbindin antibodies to determine Purkinje neuron numbers after the varioustreatments. B, The effects of p75mICD and CMV infection at 50 moi on vacuole size were quan-tified by measuring the diameters of all visible lysosensor blue-positive vacuoles in 6 –12 Pur-kinje neurons per treatment condition in at least three independent experiments. Usually, thetotal number of vacuoles measured per condition was between 100 and 200. The size distribu-tion was graphed as a percentage of total vacuoles that were within the indicated size ranges.**,*Significant difference from 25K�S (**p � 0.01; *p � 0.05).


Huntington’s disease (Kegel et al., 2000; Petersen et al., 2001),prion encephalopathies (Boellaard et al., 1991; Jeffrey et al.,1992), and diffuse Lewy body disease (Zhu et al., 2003). Extensivecytoplasmic vacuole formation, consistent with autophagy, alsohas been described in degenerating cerebellar Purkinje neuronsin models of ischemia (Fessatidis et al., 1993; Barenberg et al.,2001) and in spinocerebellar ataxia 1 transgenic mice (Skinner etal., 2001). Recently, autophagy has been implicated in the deathof cerebellar Purkinje neurons in the Lurcher mouse, which suf-fers from extensive Purkinje neuron degeneration caused by apoint mutation in the �2 glutamate receptor (Zuo et al., 1997).Two reports have also shown that Lurcher Purkinje neuronsdemonstrate ultrastructural features of autophagy at a time whenthey begin to degenerate (Yue et al., 2002; Selimi et al., 2003). Inaddition, overexpression of the mutant glutamate receptor in-duced autophagy and death in human embryonic kidney 293 cells(Yue et al., 2002). In the current study, we have shown that auto-phagy is greatly enhanced in Purkinje neurons that are dying as aresult of trophic factor deprivation, and treatments that decreaseautophagy are associated with increased survival, lending furthersupport to the hypothesis that enhanced autophagy is involved inPurkinje neuron degeneration. Moreover, we have identified arole for p75ntr as a possible mediator of autophagy and death inPurkinje neurons.

It is generally accepted that the neurotrophins NGF, BDNF,NT-3, and NT-4/5 promote survival and differentiation by bind-ing to their cognate Trk receptors (Ibanez et al., 1992). In con-trast, deciphering the role of p75ntr signaling in mediating neu-rotrophin effects has been complicated by several factors. First,p75ntr shares structural homologies with members of the tumornecrosis factor receptor family of death receptors and lacks in-trinsic tyrosine kinase activity. Second, all four neurotrophins areable to bind to p75ntr with similar affinity but display selectivitywhen binding to the Trk receptors (Klein et al., 1991a,b; Lamballeet al., 1991). Third, p75ntr is often coexpressed with Trk recep-tors, and neurotrophin effects are dependent on the relative com-plements of Trk and p75 neurotrophin receptors (for review, seeRabizadeh and Bredesen, 2003). In this context, it has beenshown that the presence of p75ntr is required for high-affinitybinding of neurotrophins to Trk receptors (Hempstead et al.,1991). Yet another layer of complexity is added when one con-siders that unprocessed proneurotrophins demonstrate higher-affinity binding to p75ntr but have negligible binding to Trkreceptors (Lee et al., 2001). Finally, p75ntr may also function as acoreceptor with the Nogo receptor (Wang et al., 2002) or as areceptor for prion (Della-Bianca et al., 2001) and �-amyloid pro-teins (Yaar et al., 1997). These complexities in neurotrophin sig-naling may provide some explanation for the paradoxical rolesascribed to p75ntr in the literature. Expression of p75ntr is capa-ble of inducing death, especially when neurotrophin concentra-tions are limited (Rabizadeh et al., 1993; Barrett and Georgiou,1996). In addition, p75ntr signaling can result in death in re-sponse to neurotrophin binding (Casaccia-Bonnefil et al., 1996;Frade et al., 1996; Kuner and Hertel, 1998; Friedman, 2000). Incontrast, other reports suggest that p75ntr plays an importantand necessary role in mediating survival in response to neurotro-phins (Barrett and Bartlett, 1994; Barrett et al., 1998; DeFreitas etal., 2001; Bui et al., 2002).

In the current report, we provide evidence that p75ntr playsan important role in mediating autophagy and death of cerebellarPurkinje neurons induced by trophic factor withdrawal. The pro-motion of survival and the reduction of autophagic vacuolationrequired high concentrations of NGF, suggesting the involve-

ment of p75ntr. TrkA phosphorylation was not correlated withthe autophagic vacuolation induced by trophic factor withdrawalor with neuroprotection mediated by NGF. Decreasing availableneurotrophin with NGF-neutralizing antibodies induced auto-phagic vacuolation and death of Purkinje neurons. Antisense top75ntr decreased autophagy and completely inhibited the loss ofPurkinje neurons in response to trophic factor withdrawal.Moreover, adenoviral-mediated overexpression of a myristoy-lated rat p75ntr intracellular domain protein (p75mICD) re-sulted in significantly increased autophagic vacuolation that wasalso accompanied by a significant loss of Purkinje neurons. Fi-nally, our results directly show that Purkinje neuron death in-duced by either trophic factor withdrawal or p75mICD overex-pression is associated with increased autophagic vacuolation,suggesting an important role for autophagy during Purkinje neu-ron degeneration.

Our results with p75ntr antisense suggest a dual role forp75ntr in mediating both the survival and death of Purkinje neu-rons. Antisense to p75ntr decreased the survival of Purkinje neu-rons maintained in healthy conditions. The loss of neurons in-duced with p75ntr antisense occurred at a time when NGFsynthesis becomes detectable, suggesting that p75ntr is requiredfor the basal survival of the cerebellar neurons that is mediated byautocrine and paracrine neurotrophin production in theses cul-tures. In contrast, Purkinje neurons pretreated with p75ntr anti-sense were completely resistant to death and demonstrated de-creased vacuolation in response to trophic factor withdrawal.Collectively, our results suggest that neurotrophin signalingthrough p75ntr can regulate the survival and death of Purkinjeneurons. One can speculate that in the presence of appropriateneurotrophin, signaling from p75ntr can promote Purkinje sur-vival, and decreasing neurotrophin levels can lead to a loss ofprosurvival p75ntr signals. However, it is also possible that a lossof appropriate neurotrophins not only leads to a loss of prosur-vival signals but also may result in the generation of prodeathsignals from p75ntr. This concept of p75ntr as a dependencereceptor has been hypothesized by others to explain the paradox-ical nature of p75ntr function (Rabizadeh et al., 2000). In addi-tion, our data have not formally ruled out the possibility thattrophic factor withdrawal results in an increase of some ligandthat promotes prodeath p75ntr signals because there is evidencein the literature that p75ntr may have other ligands or may alsofunction as a coreceptor with other proteins (Yaar et al., 1997;Della-Bianca et al., 2001; Wang et al., 2002).

Despite the evidence suggesting that autophagy is upregulatedin neurodegeneration, very little is known about the cellularmechanisms that regulate autophagy in neurons (for review, seeYuan et al., 2003). Several reports suggest that the activation ofautophagy contributes to the death of neurons (Xue et al., 1999;Uchiyama, 2001; Borsello et al., 2003; Weeks, 2003). In contrast,other studies indicate a neuroprotective role for autophagy inHuntington’s disease and �-synucleopathies (Qin et al., 2003;Webb et al., 2003). These studies demonstrate that autophagymay be a mechanism for mutant protein degradation and theprevention of plaque formation. Similarly, in cases in which mi-tochondria are extensively damaged, autophagy may be protec-tive by sequestering and degrading defective mitochondria beforethey can release death-inducing proteins (Lemasters et al., 2002;Tolkovsky et al., 2002). One can speculate that the degree andspecificity of autophagy may determine cell fate. By elucidatingthe molecular pathways that regulate autophagy, it may be pos-sible to determine which components of autophagy may contrib-ute to neurodegeneration and which may be neuroprotective. In


addition to the questions regarding autophagy as a survival ordeath mechanism, it will be of great importance to understandthe effects of autophagy on neuronal function, such as the forma-tion and maintenance of synapses, the transduction of electricalimpulses, and neurotransmitter release.

ReferencesAnglade P, Vyas S, Javoy-Agid F, Herrero MT, Michel PP, Marquez J, Mouatt-

Prigent A, Ruberg M, Hirsch EC, Agid Y (1997) Apoptosis and autoph-agy in nigral neurons of patients with Parkinson’s disease. Histol His-topathol 12:25–31.

Bailey A, Luthert P, Dean A, Harding B, Janota I, Montgomery M, Rutter M,Lantos P (1998) A clinicopathological study of autism. Brain 121:889–905.

Baptista CA, Hatten ME, Blazeski R, Mason CA (1994) Cell-cell interactionsinfluence survival and differentiation of purified Purkinje cells in vitro.Neuron 12:243–260.

Barenberg P, Strahlendorf H, Strahlendorf J (2001) Hypoxia induces anexcitotoxic-type of dark cell degeneration in cerebellar Purkinje neurons.Neurosci Res 40:245–254.

Barrett GL, Bartlett PF (1994) The p75 nerve growth factor receptor medi-ates survival or death depending on the stage of sensory neuron develop-ment. Proc Natl Acad Sci USA 91:6501– 6505.

Barrett GL, Georgiou A (1996) The low-affinity nerve growth factor recep-tor p75NGFR mediates death of PC12 cells after nerve growth factorwithdrawal. J Neurosci Res 45:117–128.

Barrett GL, Georgiou A, Reid K, Bartlett PF, Leung D (1998) Rescue of dorsalroot sensory neurons by nerve growth factor and neurotrophin-3, but notbrain-derived neurotrophic factor or neurotrophin-4, is dependent on thelevel of the p75 neurotrophin receptor. Neuroscience 85:1321–1328.

Bhave SV, Ghoda L, Hoffman PL (1999) Brain-derived neurotrophic factormediates the anti-apoptotic effect of NMDA in cerebellar granule neu-rons: signal transduction cascades and site of ethanol action. J Neurosci19:3277–3286.

Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine (MDC) is aspecific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66:3–14.

Boellaard JW, Kao M, Schlote W, Diringer H (1991) Neuronal autophagy inexperimental scrapie. Acta Neuropathol (Berl) 82:225–228.

Borghesani PR, Alt FW, Bottaro A, Davidson L, Aksoy S, Rathbun GA, Rob-erts TM, Swat W, Segal RA, Gu Y (2000) Abnormal development ofPurkinje cells and lymphocytes in Atm mutant mice. Proc Natl Acad SciUSA 97:3336 –3341.

Borsello T, Croquelois K, Hornung JP, Clarke PG (2003) N-methyl-D-aspartate-triggered neuronal death in organotypic hippocampal culturesis endocytic, autophagic and mediated by the c-Jun N-terminal kinasepathway. Eur J Neurosci 18:473– 485.

Bui NT, Konig HG, Culmsee C, Bauerbach E, Poppe M, Krieglstein J, PrehnJH (2002) p75 neurotrophin receptor is required for constitutive andNGF-induced survival signalling in PC12 cells and rat hippocampal neu-rones. J Neurochem 81:594 – 605.

Carter AR, Berry EM, Segal RA (2003) Regional expression of p75NTR con-tributes to neurotrophin regulation of cerebellar patterning. Mol CellNeurosci 22:1–13.

Casaccia-Bonnefil P, Carter BD, Dobrowsky RT, Chao MV (1996) Death ofoligodendrocytes mediated by the interaction of nerve growth factor withits receptor p75. Nature 383:716 –719.

Cataldo AM, Hamilton DJ, Barnett JL, Paskevich PA, Nixon RA (1996)Properties of the endosomal-lysosomal system in the human central ner-vous system: disturbances mark most neurons in populations at risk todegenerate in Alzheimer’s disease. J Neurosci 16:186 –199.

Cheema SS, Barrett GL, Bartlett PF (1996) Reducing p75 nerve growth fac-tor receptor levels using antisense oligonucleotides prevents the loss ofaxotomized sensory neurons in the dorsal root ganglia of newborn rats.J Neurosci Res 46:239 –245.

Cohen-Cory S, Elliott RC, Dreyfus CF, Black IB (1993) Depolarizing influ-ences increase low-affinity NGF receptor gene expression in cultured Pur-kinje neurons. Exp Neurol 119:165–175.

Condorelli DF, Dell’Albani P, Timmusk T, Mudo G, Belluardo N (1998)Differential regulation of BDNF and NT-3 mRNA levels in primary cul-tures of rat cerebellar neurons. Neurochem Int 32:87–91.

Coulson EJ, Reid K, Baca M, Shipham KA, Hulett SM, Kilpatrick TJ, Bartlett PF(2000) Chopper, a new death domain of the p75 neurotrophin receptor thatmediates rapid neuronal cell death. J Biol Chem 275:30537–30545.

DeFreitas MF, McQuillen PS, Shatz CJ (2001) A novel p75NTR signalingpathway promotes survival, not death, of immunopurified neocorticalsubplate neurons. J Neurosci 21:5121–5129.

Della-Bianca V, Rossi F, Armato U, Dal-Pra I, Costantini C, Perini G, Politi V,Della Valle G (2001) Neurotrophin p75 receptor is involved in neuronaldamage by prion peptide-(106 –126). J Biol Chem 276:38929 –38933.

D’Mello SR, Galli C, Ciotti T, Calissano P (1993) Induction of apoptosis incerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP. Proc Natl Acad Sci USA 90:10989–10993.

Favaron M, Manev RM, Rimland JM, Candeo P, Beccaro M, Manev H(1993) NMDA-stimulated expression of BDNF mRNA in cultured cere-bellar granule neurones. NeuroReport 4:1171–1174.

Ferrer I, Saracibar N, Gonzalez G (1991) Spongiform encephalopathy andmultisystemic degeneration. Neurologia 6:29 –33.

Fessatidis IT, Thomas VL, Shore DF, Hunt RH, Weller RO (1993) Neuro-pathological features of profoundly hypothermic circulatory arrest: anexperimental study in the pig. Cardiovasc Surg 1:155–160.

Frade JM, Rodriguez-Tebar A, Barde YA (1996) Induction of cell death by en-dogenous nerve growth factor through its p75 receptor. Nature 383:166–168.

Friedman WJ (2000) Neurotrophins induce death of hippocampal neuronsvia the p75 receptor. J Neurosci 20:6340 – 6346.

Gatti RA, Vinters HV (1985) Cerebellar pathology in ataxia-telangiectasia:the significance of basket cells. Kroc Found Ser 19:225–232.

Ghez C, Thach WT (2000) The cerebellum. In: Principles of neuroscience(Kandel ER, Schwartz JH, Jessell TM, eds), pp 832– 852. New York:McGraw-Hill.

Hempstead BL, Martin-Zanca D, Kaplan DR, Parada LF, Chao MV (1991)High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity NGF receptor. Nature 350:678 – 683.

Ibanez CF, Ebendal T, Barbany G, Murray-Rust J, Blundell TL, Persson H(1992) Disruption of the low affinity receptor-binding site in NGF allowsneuronal survival and differentiation by binding to the trk gene product.Cell 69:329 –341.

Jeffrey M, Scott JR, Williams A, Fraser H (1992) Ultrastructural features ofspongiform encephalopathy transmitted to mice from three species ofbovidae. Acta Neuropathol (Berl) 84:559 –569.

Jia L, Dourmashkin RR, Allen PD, Gray AB, Newland AC, Kelsey SM (1997) Inhi-bition of autophagy abrogates tumour necrosis factor alpha induced apopto-sis in human T-lymphoblastic leukaemic cells. Br J Haematol 98:673–685.

Kegel KB, Kim M, Sapp E, McIntyre C, Castano JG, Aronin N, DiFiglia M(2000) Huntingtin expression stimulates endosomal-lysosomal activity,endosome tubulation, and autophagy. J Neurosci 20:7268 –7278.

Klein R, Jing SQ, Nanduri V, O’Rourke E, Barbacid M (1991a) The trk proto-oncogene encodes a receptor for nerve growth factor. Cell 65:189–197.

Klein R, Nanduri V, Jing SA, Lamballe F, Tapley P, Bryant S, Cordon-CardoC, Jones KR, Reichardt LF, Barbacid M (1991b) The trkB tyrosine pro-tein kinase is a receptor for brain-derived neurotrophic factor andneurotrophin-3. Cell 66:395– 403.

Klionsky DJ, Emr SD (2000) Autophagy as a regulated pathway of cellulardegradation. Science 290:1717–1721.

Koeppen AH (1998) The hereditary ataxias. J Neuropathol Exp Neurol57:531–543.

Kuner P, Hertel C (1998) NGF induces apoptosis in a human neuroblastoma cellline expressing the neurotrophin receptor p75NTR. J Neurosci Res 54:465–474.

Lamballe F, Klein R, Barbacid M (1991) trkC, a new member of the trkfamily of tyrosine protein kinases, is a receptor for neurotrophin-3. Cell66:967–979.

Lasmezas CI, Deslys JP, Robain O, Jaegly A, Beringue V, Peyrin JM, Fournier JG,Hauw JJ, Rossier J, Dormont D (1997) Transmission of the BSE agent to micein the absence of detectable abnormal prion protein. Science 275:402–405.

Lee R, Kermani P, Teng KK, Hempstead BL (2001) Regulation of cell sur-vival by secreted proneurotrophins. Science 294:1945–1948.

Legrand C, Clos J (1991) Biochemical, immunocytochemical and morpho-logical evidence for an interaction between thyroid hormone and nervegrowth factor in the developing cerebellum of normal and hypothyroidrats. Dev Neurosci 13:382–396.

Leingartner A, Heisenberg CP, Kolbeck R, Thoenen H, Lindholm D (1994)Brain-derived neurotrophic factor increases neurotrophin-3 expressionin cerebellar granule neurons. J Biol Chem 269:828 – 830.

Lemasters JJ, Qian T, He L, Kim JS, Elmore SP, Cascio WE, Brenner DA (2002)Role of mitochondrial inner membrane permeabilization in necrotic celldeath, apoptosis, and autophagy. Antioxid Redox Signal 4:769–781.


Liberski PP, Gajdusek DC, Brown P (2002) How do neurons degenerate inprion diseases or transmissible spongiform encephalopathies (TSEs):neuronal autophagy revisited. Acta Neurobiol Exp (Warsz) 62:141–147.

Linseman DA, McClure ML, Bouchard RJ, Laessig TA, Ahmadi FA, Heiden-reich KA (2002a) Suppression of death receptor signaling in cerebellarPurkinje neurons protects neighboring granule neurons from apoptosisvia an insulin-like growth factor I-dependent mechanism. J Biol Chem277:24546 –24553.

Linseman DA, Phelps RA, Bouchard RJ, Le SS, Laessig TA, McClure ML,Heidenreich KA (2002b) Insulin-like growth factor-I blocks Bcl-2 inter-acting mediator of cell death (Bim) induction and intrinsic death signal-ing in cerebellar granule neurons. J Neurosci 22:9287–9297.

Linseman DA, Bartley CM, Le SS, Laessig TA, Bouchard RJ, Meintzer MK, Li M,Heidenreich KA (2003) Inactivation of the myocyte enhancer factor-2 re-pressor histone deacetylase-5 by endogenous Ca 2�/calmodulin-dependentkinase II promotes depolarization-mediated cerebellar granule neuron sur-vival. J Biol Chem 278:41472–41481.

Lowry KS, Murray SS, Coulson EJ, Epa R, Bartlett PF, Barrett G, Cheema SS(2001) Systemic administration of antisense p75(NTR) oligodeoxynucleoti-des rescues axotomised spinal motor neurons. J Neurosci Res 64:11–17.

Majdan M, Lachance C, Gloster A, Aloyz R, Zeindler C, Bamji S, Bhakar A,Belliveau D, Fawcett J, Miller FD, Barker PA (1997) Transgenic miceexpressing the intracellular domain of the p75 neurotrophin receptorundergo neuronal apoptosis. J Neurosci 17:6988 – 6998.

Marini AM, Rabin SJ, Lipsky RH, Mocchetti I (1998) Activity-dependentrelease of brain-derived neurotrophic factor underlies the neuroprotec-tive effect of N-methyl-D-aspartate. J Biol Chem 273:29394 –29399.

Martinez-Murillo R, Caro L, Nieto-Sampedro M (1993) Lesion-inducedexpression of low-affinity nerve growth factor receptor-immunoreactiveprotein in Purkinje cells of the adult rat. Neuroscience 52:587–593.

Mitchener JS, Shelburne JD, Bradford WD, Hawkins HK (1976) Cellularautophagocytosis induced by deprivation of serum and amino acids inHeLa cells. Am J Pathol 83:485– 491.

Mount HT, Elkabes S, Dreyfus CF, Black IB (1998) Differential involvementof metabotropic and p75 neurotrophin receptors in effects of nervegrowth factor and neurotrophin-3 on cultured Purkinje cell survival.J Neurochem 70:1045–1053.

Munafo DB, Colombo MI (2001) A novel assay to study autophagy: regula-tion of autophagosome vacuole size by amino acid deprivation. J Cell Sci114:3619 –3629.

Murray SS, Bartlett PF, Lopes EC, Coulson EJ, Greferath U, Cheema SS(2003) Low-affinity neurotrophin receptor with targeted mutation ofexon 3 is capable of mediating the death of axotomized neurons. Clin ExpPharmacol Physiol 30:217–222.

Nixon RA, Cataldo AM, Mathews PM (2000) The endosomal-lysosomalsystem of neurons in Alzheimer’s disease pathogenesis: a review. Neuro-chem Res 25:1161–1172.

Petersen A, Larsen KE, Behr GG, Romero N, Przedborski S, Brundin P, SulzerD (2001) Expanded CAG repeats in exon 1 of the Huntington’s diseasegene stimulate dopamine-mediated striatal neuron autophagy and de-generation. Hum Mol Genet 10:1243–1254.

Qin ZH, Wang Y, Kegel KB, Kazantsev A, Apostol BL, Thompson LM, YoderJ, Aronin N, DiFiglia M (2003) Autophagy regulates the processing ofamino terminal Huntingtin fragments. Hum Mol Genet 12:3231–3244.

Rabizadeh S, Bredesen DE (2003) Ten years on: mediation of cell death bythe common neurotrophin receptor p75(NTR). Cytokine Growth FactorRev 14:225–239.

Rabizadeh S, Oh J, Zhong LT, Yang J, Bitler CM, Butcher LL, Bredesen DE(1993) Induction of apoptosis by the low-affinity NGF receptor. Science261:345–348.

Rabizadeh S, Ye X, Sperandio S, Wang JJ, Ellerby HM, Ellerby LM, Giza C,Andrusiak RL, Frankowski H, Yaron Y, Moayeri NN, Rovelli G, Evans CJ,Butcher LL, Nolan GP, Assa-Munt N, Bredesen DE (2000) Neurotro-phin dependence domain: a domain required for the mediation of apo-ptosis by the p75 neurotrophin receptor. J Mol Neurosci 15:215–229.

Ritvo ER, Freeman BJ, Scheibel AB, Duong T, Robinson H, Guthrie D, RitvoA (1986) Lower Purkinje cell counts in the cerebella of four autisticsubjects: initial findings of the UCLA-NSAC Autopsy Research Report.Am J Psychiatry 143:862– 866.

Roux PP, Bhakar AL, Kennedy TE, Barker PA (2001) The p75 neurotrophinreceptor activates Akt (protein kinase B) through a phosphatidylinositol3-kinase-dependent pathway. J Biol Chem 276:23097–23104.

Seglen PO, Gordon PB (1982) 3-Methyladenine: specific inhibitor of auto-phagic/lysosomal protein degradation in isolated rat hepatocytes. ProcNatl Acad Sci USA 79:1889 –1892.

Selimi F, Lohof AM, Heitz S, Lalouette A, Jarvis CI, Bailly Y, Mariani J (2003)Lurcher GRID2-induced death and depolarization can be dissociated incerebellar Purkinje cells. Neuron 37:813– 819.

Shelburne JD, Arstila AU, Trump BF (1973) Studies on cellular autophago-cytosis: cyclic AMP- and dibutyryl cyclic AMP-stimulated autophagy inrat liver. Am J Pathol 72:521–540.

Skinner PJ, Vierra-Green CA, Clark HB, Zoghbi HY, Orr HT (2001) Alteredtrafficking of membrane proteins in purkinje cells of SCA1 transgenicmice. Am J Pathol 159:905–913.

Sorensen B, Tandrup T, Koltzenburg M, Jakobsen J (2003) No further lossof dorsal root ganglion cells after axotomy in p75 neurotrophin receptorknockout mice. J Comp Neurol 459:242–250.

Stromhaug PE, Klionsky DJ (2001) Approaching the molecular mechanismof autophagy. Traffic 2:524 –531.

Tolkovsky AM, Xue L, Fletcher GC, Borutaite V (2002) Mitochondrial dis-appearance from cells: a clue to the role of autophagy in programmed celldeath and disease? Biochimie 84:233–240.

Torres-Aleman I, Pons S, Arevalo MA (1994) The insulin-like growth factorI system in the rat cerebellum: developmental regulation and role in neu-ronal survival and differentiation. J Neurosci Res 39:117–126.

Uchiyama Y (2001) Autophagic cell death and its execution by lysosomalcathepsins. Arch Histol Cytol 64:233–246.

Vaudry D, Falluel-Morel A, Leuillet S, Vaudry H, Gonzalez BJ (2003) Reg-ulators of cerebellar granule cell development act through specific signal-ing pathways. Science 300:1532–1534.

Wang KC, Kim JA, Sivasankaran R, Segal R, He Z (2002) P75 interacts withthe Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature420:74 –78.

Watanabe R, Duchen LW (1993) Cerebral amyloid in human prion disease.Neuropathol Appl Neurobiol 19:253–260.

Watase K, Weeber EJ, Xu B, Antalffy B, Yuva-Paylor L, Hashimoto K, KanoM, Atkinson R, Sun Y, Armstrong DL, Sweatt JD, Orr HT, Paylor R,Zoghbi HY (2002) A long CAG repeat in the mouse Sca1 locus replicatesSCA1 features and reveals the impact of protein solubility on selectiveneurodegeneration. Neuron 34:905–919.

Webb JL, Ravikumar B, Atkins J, Skepper JN, Rubinsztein DC (2003)�-Synuclein is degraded by both autophagy and the proteasome. J BiolChem 278:25009 –25013.

Weeks JC (2003) Thinking globally, acting locally: steroid hormone regula-tion of the dendritic architecture, synaptic connectivity and death of anindividual neuron. Prog Neurobiol 70:421– 442.

Wetts R, Herrup K (1982) Interaction of granule, Purkinje and inferior oli-vary neurons in lurcher chimaeric mice. I. Qualitative studies. J EmbryolExp Morphol 68:87–98.

Xue L, Fletcher GC, Tolkovsky AM (1999) Autophagy is activated by apo-ptotic signalling in sympathetic neurons: an alternative mechanism ofdeath execution. Mol Cell Neurosci 14:180 –198.

Yaar M, Zhai S, Pilch PF, Doyle SM, Eisenhauer PB, Fine RE, Gilchrest BA(1997) Binding of beta-amyloid to the p75 neurotrophin receptor in-duces apoptosis: A possible mechanism for Alzheimer’s disease. J ClinInvest 100:2333–2340.

Yan Q, Johnson Jr EM (1988) An immunohistochemical study of the nervegrowth factor receptor in developing rats. J Neurosci 8:3481–3498.

Yuan J, Lipinski M, Degterev A (2003) Diversity in the mechanisms of neu-ronal cell death. Neuron 40:401– 413.

Yue Z, Horton A, Bravin M, DeJager PL, Selimi F, Heintz N (2002) A novelprotein complex linking the delta 2 glutamate receptor and autophagy:implications for neurodegeneration in lurcher mice. Neuron 35:921–933.

Zanjani HS, Herrup K, Guastavino JM, Delhaye-Bouchaud N, Mariani J(1994) Developmental studies of the inferior olivary nucleus in staggerermutant mice. Brain Res Dev Brain Res 82:18 –28.

Zhu JH, Guo F, Shelburne J, Watkins S, Chu CT (2003) Localization ofphosphorylated ERK/MAP kinases to mitochondria and autophago-somes in Lewy body diseases. Brain Pathol 13:473– 481.

Zuo J, De Jager PL, Takahashi KA, Jiang W, Linden DJ, Heintz N (1997)Neurodegeneration in Lurcher mice caused by mutation in delta2 gluta-mate receptor gene. Nature 388:769 –773.


cellular/molecular the p75 neurotrophin receptor …cellular/molecular the p75 neurotrophin receptor...

Documents